(A) Photoaffinity Labeling. Radioactive ethyl diazomalonyl derivatives of cysteine enzymes will be prepared and photolyzed. The resulting protein will be hydrolysed, the radioactive products separated by column chromatography, converted into the corresponding trifluoroacetyl butyl esters, and purified and identified by vpc-mass spectrometry. This methodology, if it can be established, will greatly simplify the identification of the specific amino acids attacked by carbenes in photoaffinity labeling experiments. (B) Two diazophosphonates, C6H5-CN2-PO3CH minimal over 3 and C6H5-CN2-PO equals 3, recently synthesized in our laboratory, will be tested as phosphatase inhibitors, and if successful, used in photoaffinity labeling experiments to identify the amino acids near the active sites of these enzymes. (C) Orotidine-5-phosphate decarboxylase. The new inhibitors of the enzyme, with barbiturate groups in place of the orotic acid residue, will be attached to Sephadex in an attempt to prepare affinity labeling columns for the enzyme. Experiments directed toward the determination of enzyme mechanism have been planned but await a good purification method. (D) Other studies of photoaffinity labeling, and studies of other decarboxylases (acetoacetate decarboxylase, oxaloacetate decarboxylase) are also in progress.